Low total arsenic oyster oligopeptides, preparation method and application thereof

Abstract

This paper discloses oyster oligopeptides with low total arsenic, their preparation method, and applications. To address the issue of effectively removing total arsenic during the preparation of oyster oligopeptides, the key technical points are: S100, preparing oyster enzymatic hydrolysate; S200, removing arsenic from the oyster enzymatic hydrolysate; S300, using the arsenic-removed oyster enzymatic hydrolysate to prepare oyster oligopeptide powder. The effect is that it enables the transfer of methyl groups from arsenobetaine (AsB) and their use in the synthesis of proteins and other substances, thereby converting the arsenic in arsenobetaine (AsB) from an organic state to an inorganic ionic form. The arsenic ions are then removed through processes such as electrodialysis and activated carbon adsorption, achieving an arsenic removal rate of over 90%.

Description

Technical Field

[0001] This invention belongs to the field of small molecule peptide processing, specifically relating to a low total arsenic oyster oligopeptide, its preparation method, and its application. Background Technology

[0002] Marine-derived bioactive peptides are currently a research hotspot in the industry. Oyster peptides prepared through enzymatic hydrolysis have more pronounced and effective functions than oysters themselves, such as enhancing male function, anti-fatigue, anti-tumor, and antioxidant effects. Moreover, their structure is simpler, digestion and absorption are faster and better, stability is higher, and immunogenicity is low or even non-immunogenic, showing broad application prospects. However, oysters absorb and accumulate arsenic from the marine environment. Consuming oysters with excessive arsenic content will result in some arsenic accumulating in the human body, posing potential health hazards and even causing irreversible damage. Therefore, if an arsenic removal process is not performed during the preparation of oyster oligopeptides, arsenic will accumulate in the final oyster peptide product, leading to product defects and even food safety issues.

[0003] Current research has covered heavy metal removal processes from enzymatic hydrolysates. Chinese patent application CN104970274A discloses a method for removing heavy metals from marine animal enzymatic hydrolysates, and Chinese patent application CN110833135A discloses a method for removing heavy metals from shellfish enzymatic hydrolysates. However, neither of these methods specifically targets the removal of a particular heavy metal. Furthermore, Chinese patent application CN105903447A discloses a process for adsorbing cadmium from enzymatic hydrolysates, and Chinese patent application CN105964009A discloses a process for adsorbing chromium from enzymatic hydrolysates. These are all processes for the adsorption and removal of single heavy metals, but there is no research on the removal of total arsenic.

[0004] The bioactive peptide market has broad prospects, and oyster peptides are one of the hot topics. However, there are currently no reports on the total arsenic removal process during the preparation of oyster oligopeptides. This is because GB 2762-2017, "National Food Safety Standard - Limits of Contaminants in Food," only sets limits for inorganic arsenic, not total arsenic. Currently, oyster peptide products on the market generally have low inorganic arsenic content, meeting national requirements, but high total arsenic content. On the other hand, GB16740-2014, "National Food Safety Standard - Health Food," stipulates that the total arsenic limit for general health foods is 1.0 mg / kg, and the limit for liquid and infant health foods is 0.3 mg / kg. Most oyster peptides on the market exceed these limits, thus restricting their application, promotion, and development in the health food and functional food sectors. Furthermore, long-term consumption of oyster peptides with high total arsenic content poses certain health risks. Therefore, developing a method for preparing oyster oligopeptides with low total arsenic content is of great significance and value to the bioactive peptide industry and society. Summary of the Invention

[0005] To address the problem of effectively removing total arsenic in the preparation of oyster oligopeptides, in a first aspect, a method for preparing oyster oligopeptides with low total arsenic according to some embodiments of this application includes...

[0006] S100, Preparation of oyster enzymatic hydrolysate;

[0007] S200, Remove arsenic from the oyster enzymatic hydrolysate;

[0008] S300. Oyster oligopeptide powder is prepared using the arsenic-free oyster enzymatic hydrolysate.

[0009] According to some embodiments of this application, the method for preparing low-total-arsenic oyster oligopeptides, step S1 specifically includes:

[0010] S101. After crushing and homogenizing the oysters to obtain oyster raw material, the oyster raw material is placed in an enzymatic hydrolysis tank;

[0011] S102. Add 10 to 20 times the mass volume of water to the enzymatic hydrolysis tank;

[0012] S103. Add 2-5% of the mass of the oyster raw material to the enzymatic hydrolysis tank, and enzymatically hydrolyze for 2-5 hours under the conditions of pH 7.0-9.0 and reaction temperature 35-55℃ to obtain oyster enzymatic hydrolysate.

[0013] According to some embodiments of this application, in the method for preparing low total arsenic oyster oligopeptides, in step S103, the composition and mass ratio of the complex protease is: alkaline protease: bromelain: flavor protease = (4~6): (2~4): (2~4).

[0014] According to some embodiments of this application, the method for preparing low-total-arsenic oyster oligopeptides, step S2 specifically includes:

[0015] S201. Add 0.1-0.3% of Corynebacterium glutamicum bacterial solution (by volume of oyster enzymatic hydrolysate) to the oyster enzymatic hydrolysate, react for 1-2 hours at pH 6.8-7.3 and reaction temperature 25-35℃, inactivate enzymes, sterilize, and centrifuge to obtain a clear solution.

[0016] S202. The clear liquid is introduced into the electrodialysis equipment. The first-stage electrodialysis is started. When the liquid in the container is about 50% full, the second-stage electrodialysis is started. The voltage is adjusted to 55-75V. The first-stage conductivity is controlled at 1000-1100μS / cm. When the second-stage conductivity is <500μS / cm, it is stopped. The liquid is then introduced into the storage tank through the pipeline.

[0017] S203. Heat the liquid in the storage tank to 60-70℃, add 0.5-2% activated carbon by volume of the liquid, stir for 45-80 minutes for adsorption, and then filter the liquid for decarbonization.

[0018] According to some embodiments of the present application, the preparation method of low total arsenic oyster oligopeptides includes the preparation of the Corynebacterium glutamicum bacterial culture in step S201, which involves inoculating the activated Corynebacterium glutamicum into a liquid culture medium and culturing it at pH 6.8-7.3 and a temperature of 25-35°C for 24-36 hours to obtain the Corynebacterium glutamicum bacterial culture.

[0019] According to some embodiments of the present application, the method for preparing low total arsenic oyster oligopeptides, in step S201, Corynebacterium glutamicum was purchased from the China General Microbiological Culture Collection Center (CGMCC) No. 1.816.

[0020] According to some embodiments of the present application, in the method for preparing low total arsenic oyster oligopeptides, the enzyme inactivation and sterilization in step S201 are performed by heating the liquid to 95°C and holding it for 15 minutes after the reaction is completed.

[0021] According to some embodiments of this application, the method for preparing low total arsenic oyster oligopeptides involves separating the feed solution after decarbonization in step S203 through a membrane with a molecular weight cutoff of 1000 Da, and then spray-drying the membrane solution to obtain oyster oligopeptide powder.

[0022] In a second aspect, the oyster oligopeptides according to some embodiments of this application are prepared by any one of the methods for preparing low-total-arsenic oyster oligopeptides.

[0023] In a second aspect, the application of Corynebacterium glutamicum in the preparation of oyster oligopeptides according to some embodiments of this application for the removal of arsenic.

[0024] The beneficial effects of this invention are:

[0025] (1) This invention provides a novel process for removing arsenic from oyster oligopeptides. This process utilizes a combination of microorganisms, electrodialysis, and activated carbon to convert the inorganic arsenic in oyster arsenic betaine (AsB) into ionic arsenic. The arsenic is then removed through ion migration under the electric field of electrodialysis and adsorption by activated carbon. Corynebacterium glutamicum produces arsenic methyltransferase during cultivation, which can transfer methyl groups from arsenic betaine (AsB) and use them to synthesize proteins and other substances. This converts the arsenic in arsenic betaine (AsB) from an organic state to an inorganic ionic form. The arsenic ions are then removed through electrodialysis, activated carbon adsorption, and other processes, achieving an arsenic removal rate of over 90%.

[0026] (2) This invention solves the industry pain point that the high total arsenic content of oyster peptides leads to low addition amount and poor product effect in health food, as well as the potential safety hazards of long-term consumption. It has broad application prospects and a large market. Detailed Implementation

[0027] Oysters contain relatively high levels of arsenic compounds, which are divided into inorganic and organic arsenic. Inorganic arsenic mainly consists of arsenate (AsV) and arsenite (AsIII), while organic arsenic mainly includes monomethylarsenic (MMA), dimethylarsenic (DMA), arsenobetaine (AsB), arsenic sugars, and arsenic esters. Arsenenobetaine (AsB) is the predominant form of arsenic in oysters, often accounting for up to 90% of the total arsenic content. Therefore, to obtain oyster oligopeptide powder with low total arsenic, it is necessary to remove arsenobetaine (AsB). However, arsenobetaine (AsB), with the chemical formula C5H11AsO2 and also known as 2-(trimethylarsenic)acetate, is listed as a Group 3 carcinogen in the preliminary list of carcinogens published by the International Agency for Research on Cancer (IARC) of the World Health Organization. AsB is water-soluble and cannot be directly removed during the preparation of oyster oligopeptides through physical methods such as centrifugation and filtration; moreover, as an organic compound, AsB cannot be directly removed by electrodialysis or resin column chromatography to remove heavy metal ions. This invention provides a novel arsenic removal process for oyster oligopeptides. It utilizes a combination of microorganisms, electrodialysis, and activated carbon to convert arsenic in AsB into ionic arsenic, which is then removed through ion migration under the electric field of electrodialysis and adsorption by activated carbon. To achieve the above objective, a method for preparing oyster oligopeptides with low total arsenic according to this invention includes the following steps:

[0028] S100. Preparation of oyster enzymatic hydrolysate: After cleaning, the oysters are crushed and homogenized, and placed in an enzymatic hydrolysis tank. 10-20 times the weight of water is added, and the mixture is stirred and heated. Then, 2-5% of the weight of the oysters in a compound protease is added. The pH of the solution is controlled at 7.0-9.0, and enzymatic hydrolysis is carried out at 35-55℃ for 2-5 hours to obtain the oyster enzymatic hydrolysate. Preferably, the mass ratio of the compound protease in step S1 is: neutral protease: papain: trypsin: flavor protease = (2-4): (2-3): (3-4): (1-2).

[0029] S200, removal of arsenic from the enzymatic hydrolysate:

[0030] S201. Inoculate activated Corynebacterium glutamicum into liquid culture medium, and incubate at pH 6.8–7.3 for 24–36 h at 25–35°C to obtain bacterial suspension. Preferably, the Corynebacterium glutamicum in step S2 is purchased from the China General Microbiological Culture Collection Center (CGMCC) No. 1.816.

[0031] S202. Add 0.1%–0.3% (by volume) of Corynebacterium glutamicum bacterial solution to the oyster enzymatic hydrolysate in S1, while adjusting the pH of the hydrolysate to 6.8–7.3 and the temperature to 25–35°C. React for 1–2 hours. After the reaction is complete, raise the temperature of the solution to 95°C and maintain it for 15 minutes to inactivate the enzyme and sterilize it. Then, centrifuge to obtain the clear solution.

[0032] S203. Introduce the clarified solution into the electrodialysis equipment. First, start the primary electrodialysis stage. When the solution in the container is about 50% full, start the secondary electrodialysis stage. Adjust the voltage to 55-75V, control the primary conductivity at 1000-1100μS / cm, and stop when the secondary conductivity is <500μS / cm. Then, transfer the solution through the pipeline into the storage tank.

[0033] S204. Heat the liquid in the storage tank to 60-70℃, add 0.5-2% activated carbon by volume of the liquid, stir for 45-80 minutes for adsorption, and then filter the liquid for decarbonization.

[0034] S3, Oyster oligopeptide powder: The liquid in step S204 is separated using membrane separation technology, with a molecular weight cutoff of 1000 Da, and the liquid is spray-dried to obtain oyster oligopeptide powder.

[0035] This invention provides a novel arsenic removal process for oyster oligopeptides. It utilizes a combined approach of microorganisms, electrodialysis, and activated carbon to convert the inorganic arsenic in oyster arsenic betaine (AsB) into ionic arsenic. This arsenic is then removed through ion migration under the electric field of electrodialysis and adsorption by activated carbon. *Corynebacterium glutamicum* produces arsenic methyltransferase during cultivation, which can transfer methyl groups from arsenic betaine (AsB) and use them to synthesize proteins and other substances. This transforms the arsenic in arsenic betaine (AsB) from an organic state to an inorganic ionic form, which is then removed through electrodialysis and activated carbon adsorption, achieving an arsenic removal rate of over 90%. Furthermore, this method is simple, easy to produce, and particularly effective in removing arsenic betaine, resulting in a low total arsenic content in the product. This contributes to improved food safety and greatly facilitates the promotion and application of oyster oligopeptides in functional foods and health products.

[0036] Example 1

[0037] A method for preparing oyster oligopeptides with low total arsenic includes the following steps:

[0038] S1. Preparation of Oyster Enzymatic Hydrolysate: Take 1 kg of oysters, clean them thoroughly, crush them into a homogenate, and place them in an enzymatic hydrolysis tank. Add 20 times the volume of water, stir, and heat. Then add 4% (by weight of oysters) of a compound protease. The mass ratio of the compound protease is: neutral protease: papain: trypsin: flavor protease = 3:2:3:1. Control the pH of the solution at 7.5, and enzymatically hydrolyze at 50℃ for 4 hours to obtain the oyster enzymatic hydrolysate.

[0039] S2. Removal of arsenic from the enzymatic hydrolysate:

[0040] Activated Corynebacterium glutamicum was purchased from the China General Microbiological Culture Collection Center (CGMCC) 1.816, inoculated into liquid culture medium, and cultured at pH 7.0 for 24 hours at 30°C to obtain bacterial suspension.

[0041] Add 0.1% (v / v) of Corynebacterium glutamicum solution to the oyster enzymatic hydrolysate in S1, while adjusting the pH of the hydrolysate to 7.0 and maintaining the temperature at 30°C for 1 hour. After the reaction, raise the temperature of the solution to 95°C and maintain it for 15 minutes to inactivate the enzyme and sterilize it. Then, centrifuge to obtain the clear solution.

[0042] The clarified solution is introduced into the electrodialysis equipment. First, the first-stage electrodialysis is started. When the solution in the container is about 50% full, the second-stage electrodialysis is started. The voltage is adjusted to 65V, and the first-stage conductivity is controlled at 1000μS / cm. When the second-stage conductivity is <500μS / cm, it is stopped, and the solution is transferred to the storage tank through the pipeline.

[0043] Heat the liquid in the storage tank to 70°C, add 0.5% activated carbon by volume of the liquid, stir for 45 minutes for adsorption, and then filter the liquid for decarbonization.

[0044] S3, Oyster Oligopeptide Powder: The liquid in S2 is separated using membrane separation technology, with a molecular weight cutoff of 1000 Da, and the resulting liquid is spray-dried to obtain oyster oligopeptide powder.

[0045] Example 2

[0046] A method for preparing oyster oligopeptides with low total arsenic includes the following steps:

[0047] S1. Preparation of Oyster Enzymatic Hydrolysate: Take 1 kg of oysters, clean them thoroughly, crush them into a homogenate, and place them in an enzymatic hydrolysis tank. Add 15 times the volume of water by weight, stir, and heat. Then add 3% (by weight of oysters) of a compound protease. The mass ratio of the compound protease is: neutral protease: papain: trypsin: flavor protease = 2:3:3:2. Control the pH of the solution at 8.0, and enzymatically hydrolyze at 45℃ for 3 hours to obtain the oyster enzymatic hydrolysate.

[0048] S2, Removal of arsenic from the enzymatic hydrolysate

[0049] Activated Corynebacterium glutamicum was purchased from the China General Microbiological Culture Collection Center (CGMCC) 1.816, inoculated into liquid culture medium, and cultured at pH 6.8 for 36 hours at 32°C to obtain bacterial suspension.

[0050] Add 0.2% (v / v) of Corynebacterium glutamicum solution to the oyster enzymatic hydrolysate in S1, while adjusting the pH of the hydrolysate to 6.8 and maintaining the temperature at 32°C for 2 hours. After the reaction, raise the temperature of the solution to 95°C and maintain it for 15 minutes to inactivate the enzyme and sterilize it. Then, centrifuge to obtain the clear solution.

[0051] The clarified solution is introduced into the electrodialysis equipment. First, the first-stage electrodialysis is started. When the solution in the container is about 50% full, the second-stage electrodialysis is started. The voltage is adjusted to 55V, and the first-stage conductivity is controlled at 1100μS / cm. When the second-stage conductivity is <500μS / cm, it is stopped, and the solution is transferred to the storage tank through the pipeline.

[0052] Heat the liquid in the storage tank to 60°C, add 1.0% of the liquid volume of activated carbon, stir for 80 minutes for adsorption, and then filter the liquid for decarbonization.

[0053] S3, Oyster Oligopeptide Powder: The liquid in S2 is separated using membrane separation technology, with a molecular weight cutoff of 1000 Da, and the resulting liquid is spray-dried to obtain oyster oligopeptide powder.

[0054] Example 3

[0055] A method for preparing oyster oligopeptides with low total arsenic includes the following steps:

[0056] S1. Preparation of Oyster Enzymatic Hydrolysate: Take 1 kg of oysters, clean them thoroughly, crush them into a homogenate, and place them in an enzymatic hydrolysis tank. Add 10 times the volume of water, stir, and heat. Then add 5% (by weight of oyster) of a compound protease. The mass ratio of the compound protease is: neutral protease: papain: trypsin: flavor protease = 4:2:3:2. Control the pH of the solution at 9.0, and enzymatically hydrolyze at 55℃ for 5 hours to obtain the oyster enzymatic hydrolysate.

[0057] S2. Removal of arsenic from the enzymatic hydrolysate:

[0058] Activated Corynebacterium glutamicum was purchased from the China General Microbiological Culture Collection Center (CGMCC) 1.816, inoculated into liquid culture medium, and cultured at pH 7.3 and 35°C for 28 hours to obtain bacterial suspension.

[0059] Add 0.2% (v / v) of Corynebacterium glutamicum solution to the oyster enzymatic hydrolysate in S1, while adjusting the pH of the hydrolysate to 7.3 and maintaining the temperature at 35°C for 2 hours. After the reaction, raise the temperature of the solution to 95°C and maintain it for 15 minutes to inactivate the enzyme and sterilize it. Then, centrifuge to obtain the clear solution.

[0060] The clarified solution is introduced into the electrodialysis equipment. First, the first-stage electrodialysis is started. When the solution in the container is about 50% full, the second-stage electrodialysis is started. The voltage is adjusted to 75V, and the first-stage conductivity is controlled at 1000μS / cm. When the second-stage conductivity is <500μS / cm, it is stopped, and the solution is transferred to the storage tank through the pipeline.

[0061] Heat the liquid in the storage tank to 70°C, add 1.5% activated carbon by volume of the liquid, stir for 60 minutes for adsorption, and then filter the liquid for decarbonization.

[0062] S3, Oyster Oligopeptide Powder: The liquid in S2 is separated using membrane separation technology, with a molecular weight cutoff of 1000 Da, and the resulting liquid is spray-dried to obtain oyster oligopeptide powder.

[0063] Comparative Example 1

[0064] A method for preparing oyster oligopeptides with low total arsenic, comprising:

[0065] S1. Preparation of Oyster Enzymatic Hydrolysate: Take 1 kg of oysters, clean them thoroughly, crush them into a homogenate, and place them in an enzymatic hydrolysis tank. Add 20 times the volume of water, stir, and heat. Then add 4% (by weight of oysters) of a compound protease. The mass ratio of the compound protease is: neutral protease: papain: trypsin: flavor protease = 3:2:3:1. Control the pH of the solution at 7.5, and enzymatically hydrolyze at 50℃ for 4 hours to obtain the oyster enzymatic hydrolysate.

[0066] S3. Oyster oligopeptide powder: After centrifugation of oyster enzymatic hydrolysate, the supernatant is separated using membrane separation technology with a molecular weight cutoff of 1000 Da. The resulting solution is spray-dried to obtain oyster oligopeptide powder.

[0067] The method for preparing low-total-arsenic oyster oligopeptides in Comparative Example 1 is the same as that in Example 1 except for step S2. It can be seen that Comparative Example 1 omits the step of removing arsenic from the enzymatic hydrolysate.

[0068] Comparative Example 2

[0069] A method for preparing oyster oligopeptides with low total arsenic includes the following steps:

[0070] S1. Preparation of Oyster Enzymatic Hydrolysate: Take 1 kg of oysters, clean them thoroughly, crush them into a homogenate, and place them in an enzymatic hydrolysis tank. Add 20 times the volume of water, stir, and heat. Then add 4% (by weight of oysters) of a compound protease. The mass ratio of the compound protease is: neutral protease: papain: trypsin: flavor protease = 3:2:3:1. Control the pH of the solution at 7.5, and enzymatically hydrolyze at 50℃ for 4 hours to obtain the oyster enzymatic hydrolysate.

[0071] Step S2: Removal of arsenic from the enzymatic hydrolysate

[0072] Activated Corynebacterium glutamicum was purchased from the China General Microbiological Culture Collection Center (CGMCC) 1.816, inoculated into liquid culture medium, and cultured at pH 6.8 for 36 hours at 32°C to obtain bacterial suspension.

[0073] Add 0.2% (v / v) of Corynebacterium glutamicum solution to the oyster enzymatic hydrolysate in S1, while adjusting the pH of the hydrolysate to 6.8 and maintaining the temperature at 32°C for 2 hours. After the reaction, raise the temperature of the solution to 95°C and maintain it for 15 minutes to inactivate the enzyme and sterilize it. Then, centrifuge to obtain the clear solution.

[0074] S3, Oyster Oligopeptide Powder: The supernatant in S2 is separated using membrane separation technology, with a molecular weight cutoff of 1000 Da, and the supernatant is spray-dried to obtain oyster oligopeptide powder.

[0075] The preparation method of low total arsenic oyster oligopeptides in Comparative Example 2 is different from that in Example 1 in step S2, while the other steps are the same as in Example 1. It can be seen that Comparative Example 2 omits the electrodialysis and activated carbon treatment steps.

[0076] Comparative Example 3

[0077] A method for preparing oyster oligopeptides with low total arsenic includes the following steps:

[0078] S1. Preparation of Oyster Enzymatic Hydrolysate: Take 1 kg of oysters, clean them thoroughly, crush them into a homogenate, and place them in an enzymatic hydrolysis tank. Add 20 times the volume of water, stir, and heat. Then add 4% (by weight of oysters) of a compound protease. The mass ratio of the compound protease is: neutral protease: papain: trypsin: flavor protease = 3:2:3:1. Control the pH of the solution at 7.5, and enzymatically hydrolyze at 50℃ for 4 hours to obtain the oyster enzymatic hydrolysate.

[0079] S2, Removal of arsenic from the enzymatic hydrolysate

[0080] Centrifuge the oyster enzymatic hydrolysate S1 to obtain a clear liquid. Introduce the clear liquid into an electrodialysis device. First, start the primary electrodialysis stage. When the liquid volume in the container is approximately 50%, start the secondary electrodialysis stage. Adjust the voltage to 65V, controlling the primary conductivity at 1000 μS / cm. Stop the secondary conductivity when it is <500 μS / cm, and then transfer the liquid through a pipeline into a storage tank.

[0081] Heat the liquid in the storage tank to 70°C, add 0.5% activated carbon by volume of the liquid, stir for 45 minutes for adsorption, and then filter the liquid for decarbonization.

[0082] S3, Oyster Oligopeptide Powder: The liquid in S2 is separated using membrane separation technology, with a molecular weight cutoff of 1000 Da, and the resulting liquid is spray-dried to obtain oyster oligopeptide powder.

[0083] The preparation method of low total arsenic oyster oligopeptides in Comparative Example 3 is different from that in Example 1 in step S2, while the other steps are the same as in Example 1. It can be seen that Comparative Example 3 omits the step of adding bacterial solution and only performs electrodialysis and activated carbon treatment.

[0084] Comparative Example 4

[0085] A method for preparing oyster oligopeptides with low total arsenic includes the following steps:

[0086] S1. Preparation of Oyster Enzymatic Hydrolysate: Take 1 kg of oysters, clean them thoroughly, crush them into a homogenate, and place them in an enzymatic hydrolysis tank. Add 20 times the volume of water, stir, and heat. Then add 4% (by weight of oysters) of a compound protease. The mass ratio of the compound protease is: neutral protease: papain: trypsin: flavor protease = 3:2:3:1. Control the pH of the solution at 7.5, and enzymatically hydrolyze at 50℃ for 4 hours to obtain the oyster enzymatic hydrolysate.

[0087] S2, Removal of arsenic from the enzymatic hydrolysate

[0088] Activated Corynebacterium glutamicum was purchased from the China General Microbiological Culture Collection Center (CGMCC) 1.816, inoculated into liquid culture medium, and cultured at pH 7.0 for 24 hours at 30°C to obtain bacterial suspension.

[0089] Add 0.1% (v / v) of Corynebacterium glutamicum solution to the oyster enzymatic hydrolysate in S1, while adjusting the pH of the hydrolysate to 7.0 and maintaining the temperature at 30°C for 1 hour. After the reaction, raise the temperature of the solution to 95°C and maintain it for 15 minutes to inactivate the enzyme and sterilize it. Then, centrifuge to obtain the clear solution.

[0090] The clarified solution is introduced into the electrodialysis equipment. First, the first-stage electrodialysis is started. When the solution in the container is about 50% full, the second-stage electrodialysis is started. The voltage is adjusted to 65V, and the first-stage conductivity is controlled at 1000μS / cm. When the second-stage conductivity is <500μS / cm, it is stopped, and the solution is transferred to the storage tank through the pipeline.

[0091] S3, Oyster Oligopeptide Powder: The liquid in S2 is separated using membrane separation technology, with a molecular weight cutoff of 1000 Da, and the resulting liquid is spray-dried to obtain oyster oligopeptide powder.

[0092] The preparation method of low total arsenic oyster oligopeptides in Comparative Example 4 is different from that in Example 1 in step S2, while the other steps are the same as in Example 1. It can be seen that Comparative Example 4 omits the activated carbon treatment step and only performs bacterial addition and electrodialysis.

[0093] Comparative Example 5

[0094] A method for preparing oyster oligopeptides with low total arsenic includes the following steps:

[0095] S1. Preparation of Oyster Enzymatic Hydrolysate: Take 1 kg of oysters, clean them thoroughly, crush them into a homogenate, and place them in an enzymatic hydrolysis tank. Add 20 times the volume of water, stir, and heat. Then add 4% (by weight of oysters) of a compound protease. The mass ratio of the compound protease is: neutral protease: papain: trypsin: flavor protease = 3:2:3:1. Control the pH of the solution at 7.5, and enzymatically hydrolyze at 50℃ for 4 hours to obtain the oyster enzymatic hydrolysate.

[0096] S2, Removal of arsenic from the enzymatic hydrolysate

[0097] Activated Corynebacterium glutamicum was purchased from the China General Microbiological Culture Collection Center (CGMCC) 1.816, inoculated into liquid culture medium, and cultured at pH 7.0 for 24 hours at 30°C to obtain bacterial suspension.

[0098] Add 0.1% (by volume) of Corynebacterium glutamicum solution to the oyster enzymatic hydrolysate in S1, while adjusting the pH of the hydrolysate to 7.0 and maintaining the temperature at 30°C for 1 hour. After the reaction, raise the temperature of the solution to 95°C and maintain it for 15 minutes to inactivate the enzyme and sterilize it. Then centrifuge the solution and transfer the clear liquid to the storage tank through a pipeline.

[0099] Heat the liquid in the storage tank to 70°C, add 0.5% activated carbon by volume of the liquid, stir for 45 minutes for adsorption, and then filter the liquid for decarbonization.

[0100] S3, Oyster Oligopeptide Powder: The liquid in S2 is separated using membrane separation technology, with a molecular weight cutoff of 1000 Da, and the resulting liquid is spray-dried to obtain oyster oligopeptide powder.

[0101] The preparation method of low total arsenic oyster oligopeptides in Comparative Example 5 is different from that in Example 1 in step S2, while the other steps are the same as in Example 1. It can be seen that Comparative Example 5 omits the electrodialysis treatment step and only performs bacterial solution addition and activated carbon treatment.

[0102] Result Validation

[0103] Using oyster raw materials and oyster oligopeptide products obtained in Examples 1, 2, 3 and Comparative Examples 1, 2, 3, 4, 5 of this invention as samples, the total arsenic removal effect was verified by detecting the total arsenic and inorganic arsenic content.

[0104] Test method: GB 5009.11-2014 "National Food Safety Standard - Determination of Total Arsenic and Inorganic Arsenic in Food".

[0105] Test results are shown in Tables 1 and 2.

[0106] Table 1. Arsenic Results of Oyster Raw Materials and Oyster Oligopeptide Powder Samples from Examples 1, 2, and 3

[0107]

[0108] Table 2 Arsenic Results of Oyster Oligopeptide Powder Samples in Example 1 and Comparative Examples 1, 2, 3, and 4

[0109]

[0110] in conclusion:

[0111] 1. The experimental results in Table 1 show that the preparation process of the present invention can effectively remove the total arsenic content in oyster oligopeptides, with a removal rate of over 90%.

[0112] The inorganic arsenic content of Comparative Example 2 was significantly higher than that of Comparative Example 1, and the total arsenic content of the two was similar, indicating that after treatment with Corynebacterium glutamicum liquid, the organic arsenic in the oyster enzymatic hydrolysate could be converted into inorganic arsenic. However, there was no subsequent inorganic arsenic removal process, and the total arsenic and inorganic arsenic in the product exceeded the standard.

[0113] Comparative Examples 1 and 3 showed that electrodialysis combined with activated carbon treatment could effectively remove inorganic arsenic ions from the enzymatic hydrolysate, but the removal effect on organic arsenic was very poor.

[0114] The results of Example 1 and Comparative Examples 2, 4, and 5 show that both electrodialysis and activated carbon treatment alone can effectively remove arsenic ions, but the effect is worse than the combination of the two.

[0115] Although the invention has been described through specific embodiments, those skilled in the art will understand that the invention may also encompass other embodiments within the scope of the invention as described herein.

Claims

1. A method for preparing oyster oligopeptides with low total arsenic, characterized in that, include S100, Preparation of oyster enzymatic hydrolysate; S200, Remove arsenic from the oyster enzymatic hydrolysate; S300. Oyster oligopeptide powder is prepared using the arsenic-free oyster enzymatic hydrolysate. Step S100 specifically includes: S101. After crushing and homogenizing the oysters to obtain oyster raw materials, the oyster raw materials are placed in an enzymatic hydrolysis tank; S102. Add 10 to 20 times the mass volume of water to the enzymatic hydrolysis tank; S103. Add 2-5% of the mass of the oyster raw material to the enzymatic hydrolysis tank, and enzymatically hydrolyze for 2-5 hours under the conditions of pH 7.0-9.0 and reaction temperature 35-55℃ to obtain oyster enzymatic hydrolysate. Step S200 specifically includes: S201. Add 0.1-0.3% (by volume) of Corynebacterium glutamicum bacterial solution to the oyster enzymatic hydrolysate, react for 1-2 hours at pH 6.8-7.3 and a reaction temperature of 25-35°C, inactivate the enzyme, sterilize, and centrifuge to obtain a clear liquid; used to convert arsenic in arsenic betaine (AsB) from an organic state to an inorganic ionic form; S202. The clear liquid is introduced into the electrodialysis equipment. The first-stage electrodialysis is started. When the liquid in the container is about 50% full, the second-stage electrodialysis is started. The voltage is adjusted to 55-75V. The first-stage conductivity is controlled at 1000-1100μS / cm. When the second-stage conductivity is <500μS / cm, it is stopped. The liquid is then introduced into the storage tank through the pipeline. S203. Heat the liquid in the storage tank to 60-70℃, add 0.5-2% activated carbon by volume of the liquid, stir for 45-80 minutes for adsorption, and then filter the liquid for decarbonization. The preparation of the Corynebacterium glutamicum bacterial suspension in step S201 includes inoculating the activated Corynebacterium glutamicum into a liquid culture medium and culturing it at pH 6.8–7.3 and a temperature of 25–35°C for 24–36 hours to obtain the Corynebacterium glutamicum bacterial suspension; wherein, the Corynebacterium glutamicum was purchased from the China General Microbiological Culture Collection Center (CGMCC) No. 1.

816.

2. The method for preparing low-total-arsenic oyster oligopeptides according to claim 1, characterized in that, In step S103, the composition and mass ratio of the complex protease are: alkaline protease: bromelain: flavor protease = (4~6): (2~4): (2~4).

3. The method for preparing low-total-arsenic oyster oligopeptides according to claim 1, characterized in that, In step S201, enzyme inactivation and sterilization are performed by heating the liquid to 95°C and maintaining it for 15 minutes after the reaction is completed.

4. The method for preparing low-total-arsenic oyster oligopeptides according to claim 3, characterized in that, After decarbonization in step S203, the liquid is separated by passing it through a membrane with a molecular weight cutoff of 1000 Da, and the membrane solution is spray-dried to obtain oyster oligopeptide powder.

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